，新加坡（-OF-NMRC IRG-0003-2016），和NTU-NHG老化研究资助的国家医学研究理事会（:这项工作是在部分地由教育的新加坡教育部（授予ARC9 / 15级2）资助项目格兰特ARG / 14017）。我们想表达我们的感谢和最诚挚的感谢安德鲁·阿尔珀特博士和PolyLC团队亲切与成为可能这项研究包装材料提供给我们。

1.包装长的长度阴离子交换（LAX）毛细管柱 （注意:虽然LAX列可以在家里装，因为我们在这个协议描述，LAX列也可购得，见材料与试剂的表的其他细节）。 <ol><li>暂停50毫克弱阴离子交换包装材料的在3.5mL的包装缓冲器 （ 表1）以制备淤浆。 </li><li>组装该毛细管柱（50厘米长 - 200微米内径（ID）管）的端部使用一个母对阴配合，一个套圈和阴螺母。放置屏幕1/16&quot; 1微米OD的孔尺寸的女性与阴配件内（注意:此处所用的屏幕必须具有更小的孔尺寸比包装材料的颗粒尺寸，以防止来自所述材料的泄漏柱）。 </li><li>包用在4500 PSI操作压力泵浆毛细管。 （注:打包coluMN，直到包装材料是在列的条目）可见。 </li><li>如在点1.2描述组装该毛细管柱的另一端。 </li></ol> 2.样品制备该协议概述LERLIC-MS / MS的应用程序来分析人类脑组织作为模型蛋白质组。 （注意:如果使用其它组织或蛋白质组样品中，样品制备程序应适应。） <ol><li>组织均匀化退火: 一个。洗涤脑组织（50〜100毫克）与五分钟三次1×磷酸盐缓冲溶液。 湾均质化的组织在1:1:2.5的组织/金属珠/ SDC匀浆缓冲液 （ 表2）比（W / W / V），在4℃下，在安全锁定管以最大强度使用组织匀浆5分钟。 （注:如前面在3所示SDC匀浆缓冲液可以包括蛋白酶抑制剂）中 C。离心组织匀浆，获得来回M中的前一步骤，以10,000×克，4℃10分钟，并收集上清液到新的1.5mL管中。 天。重复BC对于剩余的粒料的步骤和直到没有沉淀观察到结合上清液作为根据需要多次。 （注:如果您有重复步骤BC两倍以上，样品和珠子移动到新的安全锁管以防止在离心步骤样品的损失）。 即量化通过二喹啉甲酸测定（BCA）12所得到的匀浆的蛋白质浓度。 </li><li>脱氧胆酸钠（SDC）辅助脑匀浆的溶液内胰蛋白酶消化13。 一个。添加10mM的二硫苏糖醇（DTT）（使用表3中的解决方案5所示的原液）到所获得的匀浆以发起的蛋白质二硫键键的还原的最终浓度。 湾在浴中30分钟期间孵育匀浆预先设定为60℃。 C。添加到匀浆使用表4的解决方案7所示的储备溶液的20mM碘乙酰胺（IAA）使终浓度。 天。孵育45分钟期间含有IAA在黑暗中在室温下将组织匀浆。 即稀释脑匀浆用稀释缓冲液的两倍（ 表3的解决方案6）。 F。在37℃下30分钟期间孵育的第二还原步骤的样品。 G。添加溶解在表2中的解决方案2的测序级修饰胰蛋白酶在蛋白质与酶比率1:50（重量/重量）。 H。通过温育过夜，在30℃消化用胰蛋白酶匀浆。 一世。通过添加甲酸（FA）的0.5％最终浓度淬灭第二天酶消化。 （注:FA的加入将导致在酸性条件下SDC盐沉淀）。 学家轻轻涡旋含有SDC precipi样品大老。 ķ。通过在12,000×g下，在10分钟期间4℃离心该样品沉淀下来SDC盐。 湖收集上清液和液体转移到一个干净新管中。 （注:从SDC颗粒这一步骤的移液时小心不要再中止和/或收集盐。） 米重新溶解在SDC再溶解缓冲液 （ 表5）的SDC粒料下剧烈涡旋1分钟。 ñ。重复步骤JL两次以恢复沉淀的肽，并结合上清。 （注:参见Serra 等 2016 13上的进一步细节的SDC-协助溶液胰蛋白酶消化协议这里适配。） </li><li>胰蛋白酶的消化脱盐样品。 一个。执行使用1克C-18盒式磁带消化的样品的脱盐。 （注意:使用一个大体积盒（5毫升）的独立地蛋白得到保证的样品中剩余的SDC的盐的适当的清洁的量之前到LC-MS / MS注射）。 湾用5mL的乙腈（ACN）执行1克C-18盒式磁带的调理。 C。由空调1克C-18盒式磁带通过5mL的清理缓冲液 （ 表6），以除去任何残留的有机溶剂。 天。加载样品到1克C-18盒。 即执行3 - 5清理步骤，使用5毫升清理缓冲器的每个步骤中的1克C-18柱的样品。 F。洗脱使用5毫升的洗脱缓冲液 （ 表7）的1克C-18盒式磁带的脱盐肽。 G。在真空中干燥浓缩洗脱的肽。 C。重新构成干燥的样品中的洗脱缓冲液的200μL的终体积。 （注意:使用剧烈和长（&gt; 10分钟），涡旋，随后在超声浴超声处理之后30分钟期间以完全重新悬浮在注射缓冲液的干燥肽） 天。调整样品LERLIC-MS / MS对ANA的注射体积lyze蛋白1 3之间，以微克。 </li></ol> 3.一维LERLIC-MS / MS分离<ol><li>液相色谱条件: 流动相: A:0.1％甲酸的水（ 表8）。 B:0.1％甲酸的乙腈（ 表9） 流量:0.4微升/分钟 柱:LAX毛细管柱（50厘米长 - 为200μmID） 一个。使用下面的1200分钟梯度:95％B 40分钟，95 - 434分钟85％B，85 - 为522分钟70％B，70 - 为124分钟，35％B，35 - 3％B 45分钟，等度在5分钟的3％B，3 - 95％B历时7分钟，并保持等度在95％B 23分钟。 湾执行使用LAX毛细管如塞拉＆盖勒特-帕劳等人指出的肽的分离。使用超高压液相色谱3。 </li><li>质谱仪配置: 一个。配置扫描事件的详细信息进行数据采集交替betwe烯全傅立叶变换质谱（FT-MS）和傅里叶变换 - 串联质谱法（FT-MS / MS）使用以下参数: A.1。数据采集​​模式:正 A.2 FT-MS参数: 质量范围:350 - 2000 M / Z 分辨率60000 微扫描:1每光谱 自动增益控制:1×10 6 A.3 FT-MS / MS参数: 分段模式:高能碰撞解离（HCD） A.4质量范围:150 - 2000 M / Z A.5分辨率:30,000 A.6前N个离子:10 微扫描:1每光谱 充电状态:&gt; 2+ 隔离宽度:2达 A.7自动增益控制:1×10 6 湾执行如使用配备有纳米电喷雾离子源在1.5千伏工作轨道阱质谱仪在3所示的肽的质谱检测。 </li></ol> 4.数据分析<ol><li>执行prote在数据库检索到LERLIC-MS / MS使用获得使用特定的蛋白质组软件中的以下参数的数据:（注:进一步的细节请参见第3）。 一个。离子公差为10ppm 湾碎片离子公差:0.05沓 C。假发现率（FDR）:1％ 天。数据库:UniProt的人体数据库 即修正修改:Carbamydomethylation在半胱氨酸 F。可变修饰（如果需要由软件）:氧化（MET），脱酰胺（Asn和谷氨酰胺）。 </li><li>自信表征和异构体脱酰胺产物在LERLIC-MS / MS数据量化: 一个。从LERLIC-MS / MS数据库导出搜索结果的spreedsheet软件进行分析。 湾查找并提取脱酰胺肽及其非脱酰胺化的同行的整个列表。 C。使用所获得的肽的列表作为参考，以5ppm质量公差的提取使用一个适当的软件这些肽的离子色谱图（XICs）。 （注意:对用于XICs提取软件的详细信息，请参阅3）。 天。目测检查得到的XICs以识别所指示3的异构体产物的分离式双峰洗脱。 （注:在MS / MS鉴定的水平可以容易地发现通过在数据库中检索结果的两个不同的保留时间的存在被引导的那些肽的异构体产物。） 即从每个脱酰胺位点/肽的异构体产物的相对定量具有基于在所获得的XICs每个识别出的异构体的峰面积进行。 </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effect+of+trypsin+digestion+buffers+on+artificial+deamidation.">Evaluation of the effect of trypsin digestion buffers on artificial deamidation.</a> J Proteome Res. 14 (2), 1308-1314 (2015).</li><li>Liu, S., Moulton, K. R., Auclair, J. R., Zhou, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mildly+acidic+conditions+eliminate+deamidation+artifact+during+proteolysis:+digestion+with+endoprotease+Glu-C+at+pH+4.5.">Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.</a> Amino Acids. 48 (4), 1059-1067 (2016).</li></ol>

作者什么都没有透露。

在该视频文章，我们提出LERLIC-MS / MS 3的步骤一步协议，一种方法来进行深入的表征和准确地确定蛋白质脱酰胺和涉及在此蛋白质修饰的酶过程的程度。 LERLIC-MS / MS是基于静电排斥亲水性相互作用色谱（ERLIC）27的原则下使用的长长度（50厘米）的LAX。使用长长度列，如在我们的研究3中所示，根据它们的等电点27 ERLIC的电位最大化到单独的肽。 LERLIC-MS / MS表示一个新颖猎枪一维分离的策略，即虽然它需要一个1200分钟梯度，如3中所指示，以获得对高度复杂蛋白质组最佳的结果样品处理过程中节省了时间上的手的工作流。甲60分钟gradien吨LERLIC-MS / MS还可以实现对模型化合物用于鸟枪蛋白质组学3中的第一时间谷氨酰胺的脱酰胺亚型的最佳分离。基于这两种应用中，我们推测，通过LAX毛细管进行第二维分离可以为中间复杂样品的分析多维色谱方法下被耦合到第一维度反相层析，先前使用由我们的组10的工作流。 蛋白质脱酰胺的分析的样品制备是一个关键的一步，需要特别注意，以防止在很大的程度上1天冬酰胺残留的假象脱酰胺的发生。浩等人。发现，在温和的酸性pH胰蛋白酶消化显著防止样品制备28中假象脱酰胺幻影。我们已经在这项研究中使用了我们的SDC-协助解决试PTIC消化13，其中样品处理是在pH值为6下进行。另一方面的方法中，当pH保持低于6 29更强的酸性条件可能挑战胰蛋白酶的消化能力。解决这一问题，最近Liu 等人 。已经报道的最佳消化策略，允许上的Asn残基的脱酰胺假象的发生的成功消化和预防由在pH 4.5 30由酶Glu-C代的胰蛋白酶。刘等人报道的消化方法的实现。 30分析由LERLIC-MS / MS的Asn脱酰胺可能是需要的实验验证潜在策略。 这个视频文章中概述的协议具有很大的潜力，以进一步表征和定量定义的蛋白质脱酰胺的老化和人类疾病的参与目前未知同种型比阵列。毛皮thermore，在其它生物背景LERLIC-MS / MS的应用程序将有助于确定的潜力和蛋白质脱酰胺风险作为分子时钟修改。

脱酰胺是一种蛋白质翻译后修饰（PTM），通过天冬酰胺（ASN）和/或谷氨酰胺（Gln）残基1的变形例引入了一个负电荷的蛋白质骨架。 1比2:此修改而影响的Asn残基处的共同3生成异构产物异天冬氨酸（异Asp）和正 -天冬氨酸（ASP）。尽管如此，该比率可以通过修复酶L-isoaspartyl甲基（PIMT）3，4的干预而改变。类似地，谷氨酰胺残基的脱酰胺化，生成以预期1异构γ-谷氨酸（γ-Glu），并且的α-谷氨酸亚型（α-GLU）:7的比例3,5，但此比例可通过的作用而移普遍存在的酶的转谷氨酰胺酶2和其它转谷氨酰胺酶，转谷氨酰胺酶包括1，一个电子nzyme最近被确定为与大脑中的6外囊泡。 脱酰胺的起源可以是自发的或酶促的，前者是特别常见的谷氨酰胺残基，其中的转谷氨酰胺酶和其它酶通过转酰氨调解帧间/分子内交联（参见第3对谷氨酰胺酰氨进一步细节及其含义在几种慢性和致命人类疾病）。因此，脱酰胺是具有在所述结构上的一个关键反响和功能的影响分子4，7，8，并且需要进行了深入的化学表征3中的其不同的生物化学后果的光包括其服务老化9的作为分子时钟PTM 。 虽然天冬酰胺残留的脱酰胺已<html>相对充分表征通过自下而上鸟枪蛋白质组学1,10，谷氨酰胺残基的脱酰胺化仍然没有超出由基于电子的自由基碎片11模型化合物的具有挑战性的分析的合适表征方法。我们最近开发了一种新型一维鸟枪蛋白质组学策略（LERLIC-MS / MS）3，使在一次分析中从复杂生物样品和模型化合物谷氨酰胺和天冬酰胺脱酰胺同种型的分离。 LERLIC-MS / MS是基于使用的长长度（50厘米）离子交换柱（LAX）上静电排斥亲水性相互作用色谱（ERLIC）模式下工作的胰蛋白酶消化的肽的分离和耦合到串联质谱（LC- MS / MS）。这个新的分析策略已被用于表征和比较定量每个脱酰胺化残基在人脑组织中的程度F"&gt; 3。然而，这里概述的协议将提供旨在研究蛋白质脱酰胺的特殊性在感兴趣的生化上下文LERLIC-MS / MS的视频成像。 道德守则 本协议的所有程序已通过在新加坡南洋理工大学的机构审查委员会，并根据该机构准则已执行。

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lerlic-ms/ms for in-depth characterization and quantification of glutamine and asparagine deamidation in shotgun proteomics

蛋白质脱酰胺化的表征是势在必行破译在人体病理学和其他生化上下文该蛋白的翻译后修饰（PTM）的作用（S）和潜力。为了进行蛋白质脱酰胺的表征，我们最近开发了一种新型长条静电排斥亲水性相互作用色谱 - 串联质谱（LERLIC-MS / MS）方法，其可以分离谷氨酰胺（Gln）和天冬酰胺（ASN）同种型脱酰胺的产物从模型化合物到高度复杂的生物样品。 LERLIC-MS / MS，因此，第一鸟枪蛋白质组学策略的Gln脱酰胺同种型的分离和定量。我们还证明，作为新颖性，在这里列出的样品处理方案稳定琥珀酰亚胺中间体允许由LERLIC-MS / MS表征。如图所示这个视频文章中LERLIC-MS / MS的应用可以帮助阐明目前不明蛋白质脱酰胺的n个分子阵列。此外，LERLIC-MS / MS提供了涵盖不同的生物学背景脱酰胺酶反应的进一步理解。

谷氨酰胺和天冬酰胺残基的脱酰胺化被认为在几种慢性和致命疾病14牵连退行性蛋白修饰（DPM）。已经证明，这种PTM可以预测的抗体和其他分子的半衰期和降解状态在人体内和相似的生物学背景1，15。蛋白质脱酰胺的意义，其实超越了生物医学的背景下，因此这种修改存在于不同的蛋白质组16,17和一些研究已经证实当时脱酰胺的潜力，进行绘画的准确约会和考古遗迹18，19 。虽然意义和不同背景的蛋白质脱酰胺的功能已经肯定了半天，再是直到最近在实现这一PTM 3的深入表征方式缺乏技术进步的。 LERLIC-MS / MS 3中的应用，如在图1中所描绘，提供了一种用于在第一时间和在谷氨酰胺和天冬酰胺的单次运行解决分离脱酰胺从复杂蛋白质组或模型亚型甚至用于示出两个独立的Asn和谷氨酰胺脱酰胺那些肽化合物proteoforms。 <img alt="图1" src="/files/ftp_upload/55626/55626fig1.jpg" /> 图1:LERLIC-MS / MS的工作流图的Gln和Asp脱酰胺产物的复杂样品通过鸟枪蛋白质组学的分离和定量。 LERLIC-MS / MS涉及使用LAX毛细管柱之前的肽的一维分离用LC-MS / MS提取蛋白质和酶消化。安娜XICs裂解允许基于它们不同的酸度3，20，21在不同的保留时间洗脱的脱酰胺化的异构体产物的鉴定。 MS / MS允许脱酰胺和非脱酰胺化肽和脱酰胺化残基的信任识别之间的辨别。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55626/55626fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 显示的倒置γ/α-L-谷氨比（ 图2）转-的酶改性的中间谷氨酰胺残基可被发现并且其特征在于LERLIC-MS / MS，这可能对蛋白病的研究显著影响在神经退行性疾病3，22，23 ， </SUP&gt; 24。此外，如先前在Serra＆盖勒特-帕劳2016报道，脱酰胺化的Asn残基呈现倒置异Asp / ASP比几个未知PIMT底物蛋白也可以在人体组织由LERLIC-MS / MS鉴定3。 <img alt="图2" src="/files/ftp_upload/55626/55626fig2.jpg" /> 图2: XICs并在MS / MS级别的脱酰胺肽双保留时间的鉴定检验从发生在关键脱酰胺酶反应允许谷氨酰胺和天冬酰胺脱酰胺化的异构体和产物的鉴定的表征。 一个。示出对应于从脑蛋白DPYL2肽FQMPDQGMTSADDFFEQ＃GTK的γ/α-L-谷氨异构体的两个峰的XIC的细节二氢嘧啶相关蛋白2，反转γ/α-L-谷氨比detecteD IN那肽表示作为转谷氨酰胺酶γ谷氨酰中间体transglutamination反应的含γ谷氨酰种类的潜在意义。脱酰胺基的残余物通过MS / MS在两个保留时间证实，B。在389.1分钟对应于含有α-L-谷氨酰基肽和c。在401.4分钟对应于含有γ谷氨酰肽。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55626/55626fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 由于本文的新颖性，我们发现，LERLIC-MS / MS允许琥珀酰亚胺中间体（ 图3）的表征。样品处理过程中使用温和的酸性pH的稳定的琥珀酰亚胺改性的Asn残基25中间。因此，LERLIC-MS / MS允许succini的全蛋白质组研究酰胺中间体，不稳定和知之甚少修改与显著生理和病理的影响26。 <img alt="图3" src="/files/ftp_upload/55626/55626fig3.jpg" /> 图3: 琥珀酰亚胺中间体在天冬酰胺残基由LERLIC-MS / MS鉴定。一个。所述天冬酰胺的光谱从大脑蛋白质ALDOÇ果糖二磷酸脱酰胺肽YASICQQN＃GIVPIVEPEILPDGDHDLKR醛缩酶C.脱酰胺化残基被表示为DEAM-ASN。 湾肽YASICQQN＃GIVPIVEPEILPDGDHDLKR，在这种情况下示出了-17沓的在一个质量偏移的频谱先前脱酰胺Asn残基（SCC-ASN）由于铵损失，所述琥珀酰亚胺中间体的形成过程中发生。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55626/55626fig3large.jpg" target="_blank">请点击此处查看次的放大版。</a>是图。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 量为100毫升 </td></tr><tr><td>异丙醇</td><td> 90毫升</td></tr><tr><td>水</td><td> 10毫升</td></tr></tbody></table> 表1包装缓冲组合物（90％异丙醇）。 <table fo:keep-together.within-page="1"><tbody><tr><td colspan="2">解决方案1:乙酸铵的1M。 </td></tr><tr><td> 零件 </td><td> 量为50毫升 </td></tr><tr><td>乙酸铵</td><td> 3.86克</td></tr><tr><td>水（弥补50毫升） </td><td>约50mL </td></tr><tr><td colspan="2">解决方案2:100 mM乙酸铵的。 </td></tr><tr><td> 零件 </td><td> 量为50毫升 </strong&gt; </td></tr><tr><td>解决方法1 </td><td> 5毫升</td></tr><tr><td>水</td><td> 45毫升</td></tr><tr><td colspan="2">方案三:10％脱氧胆酸钠。 </td></tr><tr><td> 零件 </td><td> 量为1毫升 </td></tr><tr><td>脱氧胆酸钠</td><td> 0.1克</td></tr><tr><td>水</td><td> 1毫升</td></tr><tr><td colspan="2">解决方案4:SDC匀浆缓冲液（含有1％脱氧胆酸钠100 mM乙酸铵）。 </td></tr><tr><td> 零件 </td><td> 量为10毫升 </td></tr><tr><td>解决方案2 </td><td> 9毫升</td></tr><tr><td>解决方案3 </td><td> 1毫升</td></tr></tbody></table> 表2. SDC匀浆缓冲液组合物（100毫摩尔乙酸铵含有1％脱氧胆酸钠）。准备股票SOLU的乙酸铵的1 M（溶液1），并稀释和灰得到100mM乙酸铵（溶液2）。另外，制备10％的脱氧胆酸钠的储备溶液（溶液3）。如在方案4中描述制备SDC匀浆缓冲液。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2">溶液5:1 M DTT </td></tr><tr><td> 零件 </td><td> 量为10毫升 </td></tr><tr><td>二硫苏糖醇</td><td> 77毫克</td></tr><tr><td>溶液2（ 见表2） </td><td> 0.5毫升</td></tr><tr><td colspan="2">溶液6:稀释缓冲液</td></tr><tr><td> 零件 </td><td> 量为10毫升 </td></tr><tr><td>解决方案5 </td><td> 0.1毫升</td></tr><tr><td>溶液2（ 见表2） </td><td> 9.9毫升</td></tr></tbody></table>表3稀释缓冲液的组合物（100毫摩尔乙酸铵含10mM二硫苏糖醇（DTT））。制备1 M DTT的储备溶液（溶液5），并随后准备稀释缓冲液作为溶液6详述。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2">溶液7:1M的IAA </td></tr><tr><td> 零件 </td><td> 量为10毫升 </td></tr><tr><td> IAA </td><td> 93毫克</td></tr><tr><td>溶液2（ 见表2） </td><td> 0.5毫升</td></tr></tbody></table> 表4烷基化缓冲液组成（100 20mM乙酸铵含有20mM碘乙酰胺（IAA））。制备1M的IAA（溶液7）的储备溶液。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-页="总是"&gt; <tbody><tr><td> 零件 </td><td> 量为50毫升 </td></tr><tr><td>氢氧化铵</td><td> 0.25毫升</td></tr><tr><td>水</td><td> 24.75毫升</td></tr></tbody></table> 表5. SDC再溶解缓冲液组成（0.5％氢氧化铵）。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 量为100毫升 </td></tr><tr><td>三氟乙酸</td><td> 0.1毫升</td></tr><tr><td>水</td><td> 99.9毫升</td></tr></tbody></table> 表6.清理缓冲液（0.1％三氟乙酸）。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </stroNG&gt; </td><td> 量为100毫升 </td></tr><tr><td>乙腈</td><td> 75毫升</td></tr><tr><td>蚁酸</td><td> 0.1毫升</td></tr></tbody></table> 表7.洗脱缓冲液（75％乙腈，0.1％甲酸）。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 量为1000毫升 </td></tr><tr><td>蚁酸</td><td> 1毫升</td></tr><tr><td>水</td><td> 999毫升</td></tr></tbody></table> 表8流动相的组合物。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 量为1000毫升 </td></tr><tr><td>蚁酸</td> &lt;TD&gt; 1毫升</td></tr><tr><td>乙腈</td><td> 999毫升</td></tr></tbody></table> 表9.流动相B组成。

Watch this Scientific Journal Video about LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics at JoVE.com

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

这里，我们提出的长条静电排斥亲水相互作用色谱 - 串联质谱（LERLIC-MS / MS）的方法的一个步骤一步协议。这是一种新的方法，使通过鸟枪蛋白质组学的谷氨酰胺和天冬酰胺脱酰胺亚型的第一次定量和定性。

LERLIC-MS / MS进行深入表征和谷氨酰胺的定量和天冬酰胺脱酰胺在鸟枪蛋白质组学

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in ...

lerlic-msms-for-depth-characterization-quantification-glutamine

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.0K 次观看.  Nanyang Technological University. 这里，我们提出的长条静电排斥亲水相互作用色谱 - 串联质谱（LERLIC-MS / MS）的方法的一个步骤一步协议。这是一种新的方法，使通过鸟枪蛋白质组学的谷氨酰胺和天冬酰胺脱酰胺亚型的第一次定量和定性。 

视频: LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

科研

生物化学

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

SUMO 结合实体 （SUBEs） 作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

细胞和组织中辅酶A的定量

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses.

蛋白质组学设施提交和后续数据分析的TMT样品制备

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...